The broad, long-term objectives of the project is to identify and develop small molecules that inhibit the association between N-type calcium channel pore-forming subunit CaV2.2 and Mint-PDZ protein. In the previous biochemical experiments we discovered association of carboxy-terminal tail of CaV2.2 subunit with Mint1-PDZ domain and demonstrated that association of CaV2.2 with Mint1-PDZ is important for synaptic targeting and synaptic function of N-type Ca2+ channels. Based on these results we proposed that small molecule inhibitors of association between CaV2.2 carboxy-terminal and Mint1-PDZ1 can be used to treat pain. We also proposed that such molecules may serve as useful probes for studies of synaptic function of N- type Ca2+ channels. My laboratory previousy setup pilot high throughput screen (HTS) for a small molecule inhibitors of Mint-PDZ domain. The main goal of the current grant proposal is to perform a large scale HTS screen for Mint-PDZ inhibitors in collaboration with Molecular Libraries Probe Production Centers Network (MLPCN). Aim 1. In collaboration with UT Southwestern HTS core we previously developed 384-well homogeneous time-resolved fluorescence resonance energy transfer assay (HTRF) for inhibitors of association between bio- NC peptide and GST-Mint-PDZ protein. We will transfer this assay to MLPCN center and convert this assay to 1536-well format for HTS. We will provide bio-NC peptide and GST-Mint-PDZ protein to MLPCN center for screening of 300-500,000 compounds library. Aim 2. In collaboration MLPCN center we will perform a series of control HTRF experiments to filter out "false positives". A number of potential sources of "false positives" has been identified in our pilot screen and several filters were developed by our laboratory to rule them out Aim 3. We will validate the efficacy of resulting "hits" using amplified luminescent proximity homogeneous assay (ALPHA) to screen for small-molecule inhibitors of association between bio-NC and GST-Mint1-PDZ. Aim 4. We will select the "hits" that passed both HTRF and ALPHA assay for evaluation in biochemical pull- down experiments with HA-NC4 (fragment of CaV2.2 carboxy-terminal) and GST-Mint-PDZ protein. In the future generated probes will be used in studies of synaptic transmission between cultured hippocampal neurons and DRG and DH neurons. The analgesic activity of resulting "leads" will be tested in the animal models of chronic pain. The identified compounds will serve as useful probes for studies of synaptic transmission and may potentially lead to development of novel treatment for chronic pain. If successful, our study may also provide a first example for development of small molecule inhibitors of PDZ domain-mediated interactions. PUBLIC HEALTH RELEVANCE: Chronic pain (neuropathic pain, inflammatory pain, cancer pain) is a major health problem. The costs of healthcare and lost productivity due to chronic pain have been estimated around $60 billion dollars a year in the USA alone. Opiate-based drugs, such as morphine and morphine derivatives, are the primary standard of care for the treatment of chronic pain. Unfortunately, patients develop tolerance to opiates due to desensitization of the opiate receptor. Thus, alternative anti-nociceptive ("pain killing") agents need to be developed to treat chronic pain. The main goal of the proposed project is to meet this challenge by developing a small molecule that specifically targets synaptic N-type Ca2+ channels. The N-type Ca2+ channels in DRG neurons is a well validated target for chronic pain and the molecules that we propose to develop will consitute a novel and improved class of "pain-killers" for treatment of chronic pain. Generated molecules may also serve as useful probes for studies of synaptic function of N-type Ca2+ channels. [unreadable] [unreadable] [unreadable]